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Article
Takano, Hiroko ; Niimura, Sueo
Published: 新潟大学農学部研究報告 — 新潟大学農学部研究報告.  61  pp.27-33,  2008-09.  新潟大学農学部
Full Text Link: http://hdl.handle.net/10191/13064
Abstract: We previously reported in porcine oocytes that the activities of some hydroxysteroid dehydrogenases (HSDs) and the size of lipid droplets in the cytoplasm decreased as nuclear maturation progressed, and that the progression of nuclear maturation, the decrease of HSD activities and the reduction in the size of lipid droplets did not occur in those treated with olomoucine. From these results, we suggested that the changes in the steroid metabolism and the size of lipid droplets in the cytoplasm were closely associated with nuclear maturation. However, whether olomoucine directly acts on the cytoplasm to inhibit such changes could not be determined. In the present investigation, the activities of some HSDs and Sudanophilic lipid droplets were histochemically demonstrated in porcine oocytes in which the nuclear maturation was suppressed and the high cAMP level in the cytoplasm was maintained by the treatment of IBMX or dbcAMP, in order to clarify the relationship between nuclear maturation and the metabolism of steroids and the number of lipid droplets in the cytoplasm. Of the oocytes cultured with IBMX or dbcAMP for 22 hrs, 97 and 100% were in the germinal vesicle (GV) stage, respectively. The percentages of oocytes in the GV stage were significantly higher in both the treated oocytes than in control oocytes. The rates of the treated oocytes showing the activities of Δ^<5>-3β-HSD (using pregnenolone and 17α-hydroxypregnenolone as the substrates), 17β-HSD (estradiol-17β), 20α-HSD (20α-hydroxyprogesterone) and 20β-HSD (17β-hydroxyprogesterone) did not differ from those of control oocytes. Also, there were no differences in the number of lipid droplets of different sizes between IBMX- or dbcAMP-treated oocytes and control oocytes. From these findings, it was suggested that the changes in the steroid metabolism and the size of lipid droplets in the cytoplasm with oocyte maturation depend on the cAMP level in their cytoplasm rather than the progression of nuclear maturation.<br />我々は、ブタ卵母細胞において、核の成熟に伴って細胞質ではいくつかのステロイドの代謝低下と脂質小滴の小型化が起こることを以前報告した。また、核の成熟をオロモウシンで処置して阻止した卵母細胞では、ステロイド代謝の低下および脂質小滴の小型化はみられなかったことから、細胞質でのこれらの変化は核の成熟と密接に関係していることを推察して報告した。しかし、オロモウシンがステロイド代謝の低下および脂質小滴の小型化に直接関係しているのか否かは明らかにできなかった。そこで、オロモウシンとは異なった機構、すなわち細胞質のcAMPレベルを高く維持することによって核の成熟を抑制する作用のあるIBMXとdbcAMPを用いて、ブタの卵母細胞を処置し、ステロイド代謝と脂質小滴の数を組織化学的に観察した。IBMXあるいはdbcAMPで22時間処置した卵母細胞において、核は97および100%で卵核胞期にあり、これらの処置により成熟分裂の再開が抑制されていることが確認された。一方、IBMX あるいはdbcAMPで22時間処置した卵母細胞において、Δ^<5>-3β-HSD活性(基質としてpregnenoloneと17α-hydroxypregnenolone を使用)、17β-HSD 活性(estradiol-17β)、20α-HSD 活性(20α-hydroxyprogesterone)および20β-HSD活性(17β-hydroxyprogesterone)を有するものの割合、ならびに各種大きさの脂質小滴の数は、どちらも対照の卵母細胞におけるそれらと相違なかった。以上の結果から、細胞質におけるステロイド代謝の低下および脂質小滴の小型化は、核の成熟の進行よりも、細胞質のcAMP の量に依存して起こることが推察された。 Read more
2.

Article

Article
新村, 末雄 ; 大樌, 香苗
Published: 新潟大学農学部研究報告 — 新潟大学農学部研究報告.  58  pp.39-43,  2005-08.  新潟大学農学部
Full Text Link: http://hdl.handle.net/10191/657
Abstract: 体外で成熟過程にあるブタの卵母細胞、1細胞期から拡張胚盤胞期までの体外受精に由来するブタの卵子と初期胚について、グルコース-6-リン酸脱水素酵素(G-6-PDH)の活性を組織化学的に検出した。G-6-PDH活性は、胞状卵胞から採取直後の卵母 細胞では強く、この強い活性は培養後44時間の卵母細胞まで維持された。また、この酵素の活性は、媒精後の受精卵子でも強かったが、2細胞期から16細胞期の胚ではやや弱まり、弱度ないし強度となった。桑実胚期以降、酵素活性はさらに弱まり、活性を示さない胚も出現するとともに、拡張胚盤胞では活性はまったくみられなかった。以上の結果と従来のステロイド代謝能の結果とを考え合わせると、成熟過程にあるブタの卵母細胞と発生過程にあるブタの初期胚は、G-6-PDHの作用によって産生したNADPHをステロイドの生合成のために利用していることが推察された。The activity of gucose-6-phosphate dehydrogenase (G-6-PDH) in porcine oocytes and embryos was histochemically examined by the Rudolph and Klein method. Strong activity of G-6-PDH was observed in oocytes cultured for maturation and in fertilized oocytes, and blue diformazan granules produced by the enzyme reaction were spread evenly throughout the cytoplasm. In embryos, the activity somewhat decreased at the 2-cell stage, and the such activity was maintained up to the 16-cell stage. The activity weakened in embryos at the morula and early blastocyst stages, and some of them showed no enzyme activity. The enzyme activity completely disappeared from expanded blastocysts. In cleaved embryos, diformazan granules were distributed throughout the cytoplasm of blastomeres, while the amount of the granules differed among blastomeres. In early blastocysts, the granules were distributed in the cytoplasm of inner-cell-mass cells, but not in the cytoplasm of trophoblasts. The results obtained from this investigation and former studies concerning steroid metabolism seem to suggest that porcine oocytes and preimplantation embryos utilize NADPH produced by G-6-PDH for biosynthesis of steroids. Read more
3.

Article

Article
Niimura, Sueo ; Kawakami, Shin-ya
Published: 新潟大学農学部研究報告 — 新潟大学農学部研究報告.  55  pp.27-34,  2002-08.  新潟大学農学部
Full Text Link: http://hdl.handle.net/10191/25111
Abstract: The activities of hydroxysteroid dehydrogenases (HSDs) were histochemically demonstrated in mouse oocytes in the process of maturation in vivo and in vitro, and the changes in steroid metabolism during meiotic maturation were examined. In mouse oocytes soon after collection, the activities of △5-3β- HSD, 17β-HSD and 20β-HSD were observed in 87 to 97% of those, while the activity of 20α-HSD was not. The percentages of oocytes showing the activities of △5-3β-HSD (using DHA, pregnenolone and 17α- hydroxypregnenolone as the substrates), 17β -HSD (estradiol-17β and testosterone) and 20β -HSD (17α- hydroxyprogesterone and 20β-hydroxyprogesterone) did not change during maturation in vivo and in vitro. The oocytes with the activity of 20α-HSD (20α hydroxyprogesterone) appeared 4 hrs after the hCG injection or after culturing for 4 hrs and the rates of those reached 92 and l00%, respectively, 14 hrs after the hCG injection or after cultunng for 14 hrs. In the oocytes cultured for 8 hrs with olomoucine, an inhibitor of cychn-dependent kmase, nuclei were almost in the germinal vesicle stage, and the activity of 20α-HSD (20α hydroxyprogesterone) was observed in 84% of the treated oocytes. On the other hand, 81% of control oocytes also showed such the HSD activity, showing no difference from the rate of the treated oocytes. From the present findings, it was suggested that the metabolic abilities of progesterone, Viahydroxyprogesterone, 17α,20β dihydroxyprogesterone, 20β -hydroxyprogesterone, estradiol-17/7 and androgen are constantly present in mouse oocytes in the process of maturation in vivo and in vitlo. And it was also suggested that the metabolic ability of 20α-hydroxyprogesterone in mouse oocytes varies with maturation, while the change in the metabolic ability of such steroid is not related to nuclear maturation.<br />体内および体外で成熟過掛こあるマウス卵母細胞について、各種ヒドロキシステロイド脱水索酵素(HSD)の活性を組織化学的に検出し、卵母細胞の成熟に伴うステロイド代謝能の変化を検肘した。採取直後の卵母細胞において、 87ないし97%で△^5-3β-HSD、 17β-HSDおよび20β-HSDの活性が検出されたが、 20α-HSDの活性は検出されなかった。一方、 △^5-3β-HSD(基質としてDHA、 pregnenoloneおよび17α hydroxypregnenoloneを使用)、 17β-HSD (estradiol-17βとtestosterone)および20β-HSD O7α hydroxyprogesteroneと20β-hydroxyprogesterone)の活性を示す卵母細胞の割合は、体内および体外での成熟の経過に伴う変化を示さなかったが、成熟に伴って、20α-HSD (20α-hydroxyprogesterone)の活性を示す卵母細胞は出現し、その割合は高くなった。また、 8時間オロモウシン処置した卵母細胞において、核はほとんどで卵核胞期にあるとともに、 20α-HSD (20α-hydroxyprogesterone)の活性を示すものの割合は84%であり、オロモウシン処置していない対照の卵母細胞の81%と相違なかった。以上の結果から、マウス卵母細胞のprogesterone、 17α-hydroxyprogesterone、 17α,20β-dihydroxyprogesterone、 20β-hydroxyprogesterone、 estradiol-17βおよびandrogenの代謝能は、体内および体外での成熟に関わらず変化しないことが示唆された。また、マウス卵母細胞では、成熟に伴って20α- hydroxyprogesteroneの代謝能は出果して高まるが、この代謝能の変化は、核の成熟とは関係ないことが推察された。 Read more